PROJECT SUMMARY New advances in immunotherapy for cancer, based on decades of fundamental research on the regulation of T cells, have yielded tremendous optimism that even aggressive and fatal cancers, including metastatic melanoma, can one day be cured in the vast majority of patients. However, current approaches benefit only a subset of patients, and new insights into immune processes are urgently needed to define the means to improve therapies and achieve clinical benefits for a variety of different cancers. We recently discovered that the adhesion receptor, PSGL-1 (P-selectin glycoprotein ligand-1), is a new checkpoint inhibitor of T responses to chronic viral infection and a potent regulator of anti-tumor responses. The goal of this project is to determine if targeting PSGL-1 (Selplg) in tumor-specific CD8+ and/or CD4+ T cells represents a strategy to mitigate tolerance of melanoma tumors when used for adoptive cell therapy alone and when combined with additional approaches to alleviate immune suppression. Our studies showed that genetic deletion of PSGL-1 led to downregulation of multiple inhibitory receptors that are hallmarks of dysfunctional or ?exhausted? T cells including PD-1, LAG3, CD160, BTLA and TIM3, indicating that PSGL-1 modulates expression of other checkpoint inhibitors. PSGL-1-deficiency did not alter T cell migration into tissues, but rather enabled CD8+ and CD4+ T cells to mount much greater effector responses due to enhanced survival of multifunctional T cells that mediated effective anti-viral and anti-tumor responses. Our results demonstrate that this receptor may be a novel target with the potential to reverse immune suppression in patients whose cancers are unresponsive or have suboptimal responses to other immunotherapies. In mice, PSGL-1- deficiency led to dramatic control of melanoma tumor growth that was coupled with greater numbers of tumor infiltrating, multifunctional CD8+ and CD4+ T cells that expressed lower levels of PD-1. However, when tumor- specific CD8+ T cells were used to treat tumor-bearing mice by adoptive transfer, tumor control was not sustained, indicating loss of T cell anti-tumor response. In this application, we will test the hypothesis that genetic deletion of PSGL-1 in tumor-specific CD4+ and CD8+ T cells used for adoptive cell therapy, particulalry in combination, can dramatically enhance anti-tumor responses (Aim 1), but their functionality and preservation can be optimized when other mechanisms underlying T cell exhaustion are concurrently inhibited (Aim 2). We will also address whether TCR ?engineered? T cells can form memory and whether checkpoint blockade is advantageous or deleterious for memory T cell induction.